A novel noninvasive second-harmonic-generation tomographic method of mapping the structure of animal tissues by use of 100-fs laser pulses at 625nm is described. Subsurface structures were measured with this approach, which is potentially a symmetry-sensitive tool for optical histological reconstruction.

Fluorescence spectra measured from biological samples, such as tissues or cell suspensions, are usually distorted due to the light absorption by intrinsic chromophores. These distortions are aggravated by strong scattering of light inside the samples. A new method is described for a fast correction of these spectral distortions, using only steady-state spectroscopic measurements. The method is based on the formulas derived from a simplified photon diffusion model, in the isotropic one-dimensional approximation applied to a semi-infinite, highly scattering, and moderately absorbing medium with a refractive-index-matched boundary. The formulas describe the spectral distortions of the fluorescence emission and excitation spectra, together with the diffuse reflectance spectrum, as the functions of one spectral characteristic of the medium, the darkness, which is the ratio of absorption coefficient and reduced scattering coefficient. The algorithm does not involve any iterative procedures, and offers a direct, simple, and fast method for real-time spectral correction. The true fluorescence emission or excitation spectrum is directly calculated from a pair of experimental spectra: the fluorescence emission or excitation spectrum and the diffuse reflectance spectrum, measured from the same position on a sample. The correction produces the profile of the true fluorescence spectrum, the same as the one measured from the corresponding sample with an infinitely low absorption and no scattering. The restoration of the spectral profiles of true fluorescence emission and excitation spectra was tested experimentally, using highly scattering phantoms with a fluorescent dye and a deliberately added nonfluorescent dye producing strong inner-filter distortions. © 1998 Society of Photo-Optical Instrumentation Engineers.

We have measured UV fluorescence excited through two-photon absorption from native chicken tissue, using 600-nm, 500-fs pulses from a R6G dye laser. The observed emission signal was found to depend quadratically on the excitation intensity. The two-photon excitation-induced fluorescence spectrum is attributed to tryptophan residues in proteins.

We report on an analysis ofdiffuse reflectance spectra measured in conjunction with the fluorescence from normal human breast tissues and malignant breast tumors. The diffuse reflectance spectra from excised, air-equilibrated, human breast tissue samples show lower fractions of oxygenated hemoglobin and higher content offerric (Fe3) heme in malignant breast tumor samples than in normal breast tissues. Normaltissues are found to be easily deoxygenated and reoxygenated, but malignant tumors usually do not change their state as much. An analysis oftissue oxygenation parameters will be discussed with respect to an enhancement ofpredictive power offluorescence diagnostic method. The oxygenation state of tissues may be used as an additional marker in cancer diagnostics.

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Second harmonic and two photon fluorescence generated by focused 100 fs ultrashort laser pulses were used to image subsurface structures of highly scattering biological tissues. By scanning the focal point of the incident laser beam, 2-D sectional images ofthe tissue subsurface structure properties, such as symmetry and molecular compositions are obtained.

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